Figure 9.

HAN and GATA3 Family Proteins Interact in Yeast Two-Hybrid Assays and in BiFC.

(A) Yeast two-hybrid assays. Bait constructs express HAN fused with the GAL4 DNA binding domain (DBD). Prey constructs express HAN, HANL2, GNC, or GNL fused with the GAL4 activation domain (AD). Empty prey constructs expressing the GAL4 activation domain alone serve as a negative control. Left panel shows yeast patches expressing both constructs derived from independent transformed colonies, which were streaked onto SD-Leu-Trp selection plates. Right panel indicates the X-Gal–based colony lift yeast two-hybrid assay. Blue color indicates the cumulative β-galactosidase activity caused by the activation of the lacZ reporter gene, which is activated by the physical interaction between HAN and GATA3 family proteins. At least two independent experiments were performed, and the result of one representative experiment is shown.

(B) BiFC interactions between HAN and GATA3 family proteins in transiently transformed Nicotiana benthamiana leaves. For each picture, a positive interaction is indicated by GFP fluorescence (green) in nuclei (left panel), the tobacco cells are indicated by chlorophyll autofluorescence (red) (middle panel), and the two merged channels are also shown (right panel). The label HAN-GFPN represents HAN fused with the N-terminal half of GFP in frame, with similar labels used for the other constructs. Representative images of different combinations, including HAN-GFPN with HAN-GFPC, HANL2-GFPN with HAN-GFPC, GNC-GFPN with HAN-GFPC, GNL-GFPN with HAN-GFPC, FAMA-GFPC with HAN-GFPC (negative control), and FAMA-GFPN with FAMA-GFPC (positive control) (Ohashi-Ito and Bergmann, 2006), are shown. All pictures were taken using the same settings, and each interaction was confirmed three times with independent infiltrations. Bars = 20 µm.